Maltohexaose and maltoheptaose-forming amylase, and its preparation and uses

ABSTRACT

Disclosed is a novel amylase which mainly forms maltohexaose and maltoheptaose when acts on starch, but does not substantially hydrolyze maltohexaose and a lower molecular oligosaccharide than maltohexaose. The amylase can be prepared from microorganisms of the genus Alcaligenes, and has a relatively-high optimum temperature and thermal stability, as well as a relatively-wide range of optimum pH and pH stability. By using the amylase, saccharide compositions rich in maltohexaose and/or maltoheptaose or those rich in maltohexaitol and/or maltoheptaitol can be readily prepared in an industrial scale, and the saccharide compositions thus obtained can be used in a variety of food products, cosmetics and pharmaceuticals.

This is a division of parent application Ser. No. 08/526,082, filed Sep.11, 1995, now U.S. Pat. No. 5,739,024, itself a division of its parentapplication Ser. No. 08/396,746, filed Mar. 1, 1995, now U.S. Pat. No.5,527,699.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a novel maltohexaose andmaltoheptaose-forming amylase, and preparation and uses thereof, moreparticularly, to a maltohexaose and maltoheptaose-forming amylase whichmainly forms maltohexaose and maltoheptaose from starch (designated as"AMYLASE" hereinafter) and preparation thereof, as well asmicroorganisms which produce such an amylase, saccharide compositionscontaining maltohexaose and/or maltoheptaose produced therewith or thosecontaining maltohexaitol and/or maltoheptaitol obtainable byhydrogenating the maltohexaose and maltoheptaose, and compositionscontaining these saccharides.

2. Description of the Prior Art

Methods for specifically producing maltooligosaccharides such as maltoseand maltotetraose by allowing specific amylases to act on starch havebeen employed in an industrial scale, and widely used in processes ofcompositions such as foods and pharmaceuticals. Demands for saccharidescontaining a relatively-large quantity of maltooligosaccharides such asmaltohexaose and maltoheptaose have been increased because, amongmaltooligosaccharides, those with a relatively-low molecular weight areconsiderably-low in sweetness, readily digestible and absorbable.

Enzymes or amylases, derived from microorganisms, which produce arelatively-large amount of maltohexaose when act on starch are known,but no amylase which forms maltooligosaccharides higher thanmaltoheptaose has been known.

Amylases which mainly produce maltohexaose from starch can be roughlyclassified into two groups based on their actions. The one is anexo-type maltohexaose-forming amylase which hydrolyzes amylaceoussubstances so as to remove successive maltohexaose units from thenon-reducing chain ends, namely, maltohexaohydrolase (EC 3.2.1.98), andthe other is an endo-type maltohexaose-forming amylase, namely,endo-type α-amylase (EC 3.2.1.1) which acts on internal amylaceoussaccharide chains to produce maltohexaose in a relatively-largequantity.

Kainuma et al. reported in FEBS Letters, Vol.26, pp.281-285 (1972) amicroorganism of the species Aerobacter aerogenes which intracellularlyforms an exo-type maltohexaose-forming amylase which is characteristicof its relatively-low optimum temperature and thermal stability, but itis insufficient in heat tolerance for an industrial use.

J. F. Kennedy et al. reported in Starch, Vol.31, pp.235-241 (1979) amicroorganism of the species Bacillus subtilis forms an endo-typemaltohexaose-forming amylase, and Takasaki reported in Agricultural andBiological Chemistry, Vol.46, pp.1539-1547 (1982) a microorganisms ofBacillus circulans G-6 forms such an amylase. Taniguchi reported inDenpun Kagaku (Starch Science), Vol.29, pp.107-116 (1982) amicroorganism of the species Bacillus circulans F-2 forms such anamylase, and Hayashi et al. reported in Agricultural and BiologicalChemistry, Vol.52, pp.443-448 (1988) a microorganism of the speciesBacillus sp. H-167 forms such an amylase. It has been known that, amongthese amylases produced from such microorganisms, those from Bacilluscirculans G-6, Bacillus circulans F-2 and Bacillus sp. H-167 formmaltohexaose from starch in the maximum yield of about 25-30 w/w %, on adry solid basis (d.s.b.), (the wording "w/w %" is designated as "%"hereinafter, if not specified otherwise), but they do not formmaltoheptaose, and further they hydrolyze maltohexaose into maltose andmaltotetraose as their enzymatic reactions proceed.

Now referring to amylases derived from microorganisms of the speciesBacillus subtilis, they only form about 25% maltohexaose from starch anddo not hydrolyze maltohexaose. Such amylases are not suitable for anindustrial-scale production of high maltohexaose content saccharidesbecause they do not form maltohexaose in a satisfactorily-high yieldwhile forming a relatively-large quantity of lower molecularoligosaccharides under starch saccharification conditions, and becausethey could not be readily obtained in quantity from microorganisms.

In Agricultural and Biological Chemistry, Vol.42, pp.259-267 (1978)reported that a purified α-amylase, prepared from malts, mainly formsmaltohexaose and maltoheptaose during the early stage of the enzymaticreaction when acts on starch. It is also reported that the formedmaltohexaose and maltoheptaose are decomposed almost completely intolower molecular oligosaccharides such as maltose and maltotetraose asthe enzymatic reaction proceeds, and, therefore, it hardly producessaccharified products, mainly containing maltohexaose and maltoheptaose,from starch.

SUMMARY OF THE INVENTION

The present inventors pursued a novel amylase which mainly formsmaltohexaose and maltoheptaose from starch and screened microorganismswhich form such an amylase and have overcome the object. As a result,they found that a novel microorganism of the genus Alcaligenes orAlcaligenes latus D2271, isolated from a soil nearby Komagatake inYamanashi, Japan, forms a novel amylase which forms a relatively-largequantity of maltohexaose and maltoheptaose from starch (sometimesdesignated as "AMYLASE" in the specification), established thepreparation of saccharide compositions containing maltohexaose and/ormaltoheptaose by allowing the amylase to act on amylaceous substances,and also established compositions such as food products, cosmetics,pharmaceuticals and shaped products. Thus, they accomplished thisinvention.

BRIEF EXPLANATION OF THE ACCOMPANYING DRAWINGS

FIG. 1 illustrates the influence of temperature on the activity of thepresent AMYLASE.

FIG. 2 illustrates the influence of pH on the activity of the presentAMYLASE.

FIG. 3 illustrates the influence of temperature on the stability of thepresent AMYLASE.

FIG. 4 illustrates the influence of pH on the stability of the presentAMYLASE.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is to provide a novel amylase which forms arelatively-large quantity of maltohexaose and maltoheptaose from starchbut does not substantially decompose the formed maltohexaose andmaltoheptaose, and to preparations of saccharide compositions containingmaltohexaose and/or maltoheptaose by using the amylase, as well as touses of the saccharide compositions.

The results of an identification test of the novel microorganismAlcaligenes latus D2271 according to the invention are as shown in thebelow. The test was carried out according to the method as described inBiseibutsu-no-Bunrui-to-Dotei (Classification and Identification ofMicroorganisms), edited by Takeharu Hasegawa, published by GakkaiPublishing Center, Tokyo, Japan (1985).

A. Morphology

(1) Characteristics of cells formed when incubated at 27° C. in agarwith potato dextrose

Existing in a bacillus form with a size of 0.7-1.3×1.3-2.4 μm, andusually existing in a single form, but uncommonly existing in a coupled-or linked-form;

Possessing motility, asporogenicity and flagellum;

Non-acid fast;

Gram stain: Negative;

Accumulating poly-β-hydroxy butyrate;

(2) Characteristics of cells formed when incubated at 37° C. in agarwith yeast extract and malt extract

Existing in a size of about 0.5-1.0×1.3-2.4 μm after 24-hour incubation,and in a size of about 0.5-1.0×1.0-2.5 μm after 72-hour incubation;

Existing in a single form;

B. Cultural property

(1) Characteristics of colony formed when incubated at 37° C. in agarplate with potato dextrose

Shape: Circular colony having a diameter of about 1 mm after 24-hourincubation, and about 3-4 mm after 96-hour incubation;

Rim: Entire;

Projection: Umbilicate;

Gloss: Dull;

Surface: Creases-like; and

Color: Opaque, white or ocher;

(2) Characteristics of colony formed when incubated at 37° C. in agarplate with yeast extract and malt extract

Shape: Circular colony having a diameter of about 3-5 mm after 96-hourincubation;

Rim: Entire;

Projection: Half-lens type;

Gloss: Glistening;

Surface: Smooth; and

Color: White or creamy;

(3) Characteristics of colony formed when incubated at 37° C. in slantagar with potato dextrose

Growth: Satisfactory;

Shape: Thread-like;

(4) Characteristics of colony formed when stab cultured at 37° C. ingelatin plate with yeast extract and malt extract

Liquefied;

C. Physiological properties

(1) Reduction of nitrate: Positive in succinate medium;

(2) Denitrification reaction: Negative;

(3) Methyl red test: Negative;

(4) VP-test: Negative;

(5) Formation of indole: Negative;

(6) Formation of hydrogen sulfide: Negative;

(7) Hydrolysis of starch: Positive;

(8) Utilization of citric acid: Negative;

(9) Utilization of inorganic nitrogen source: Utilizing ammonium saltsand nitrates;

(10) Formation of pigment: Negative;

(11) Urease: Positive;

(12) Oxidase: Positive;

(13) Catalase: Positive;

(14) Growth conditions: Growing at a pH in the range of 5-8 and atemperature in the range of 10-41° C.;

(15) Oxygen requirements: Aerobic;

(16) Utilization of carbon source and acid formation:

    ______________________________________                                        Carbon source  Utilization                                                                            Acid formation                                        ______________________________________                                        D-Glucose      +        -                                                     D-Galactose    +        -                                                     D-Mannose      +        -                                                     D-Fructose     +        -                                                     L-Arabinose    +        -                                                     D-Xylose       +        -                                                     L-Rhamnose     +        -                                                     Maltose        +        -                                                     Sucrose        +        -                                                     Lactose        +        -                                                     Trehalose      +        -                                                     Raffinose      +        -                                                     Mannitol       -        -                                                     Dextrin        +        -                                                     Dulcitol       -        -                                                     ______________________________________                                    

(17) Decarboxylase test on amino acid: Negative against L-lysine,L-arginine and L-ornithine;

(18) DNase: Negative;

(19) Formation of 3-ketolactose: Negative; and

(20) Mol % guanine (G) plus cytosine (C) of DNA: 67%.

The bacteriological properties were compared with those of knownmicroorganisms with reference to Bergey's Manual of SystematicBacteriology, Vol.1 (1984). As a result, it was revealed that themicroorganism was identified as a microorganism of the speciesAlcaligenes latus.

Based on these results, the present inventors named this microorganism"Alcaligenes latus D2271", and deposited it on Feb. 23, 1994, inFermentation Research Institute, Agency of Industrial Science andTechnology, Ibaraki, Japan.

The deposition of the microorganism was accepted on the same day and hasbeen maintained by the institute under the accession number of FERMBP-4578.

In addition to the above-identified microorganism, other strains of thespecies Alcaligenes latus and their mutants can be suitably used in theinvention as long as they produce the present AMYLASE.

Any nutrient culture medium can be used in the invention as long asthese microorganisms can grow therein and produce the present AMYLASE,and therefore synthetic- and natural-nutrient culture media can be usedas a nutrient culture medium. Any carbon-containing substance can beused in the invention as a carbon source as long as it is utilized bythe microorganisms: Examples of such carbon sources are saccharides suchas maltose, dextrin and starch; and natural substances ofsaccharides-containing substances such as theriac and yeast extract. Theconcentration of these carbon sources used in nutrient culture media areappropriately chosen dependently on the type of carbon resources used.For example, preferable concentrations of starch used in media areusually 20% or lower, more particularly, 5% or lower, d.s.b., in view ofthe growth of microorganisms. The nitrogen sources usable in theinvention are, for example, inorganic nitrogen compounds such asammonium salts and nitrates; and organic nitrogen-containing substancessuch as urea, corn steep liquor, casein, peptone, yeast extract and beefextract. The inorganic ingredients usable in the invention are, forexample, calcium salts, magnesium salts, potassium salts, sodium salts,phosphates and other salts of manganese, zinc, iron, copper, molybdenumand cobalt.

The microorganisms used in the invention are cultured under aerobicconditions at a temperature, usually, in the range of 10-40° C.,preferably, in the range of 25-37°C., and at a pH in the range of 5-8,preferably, in the range of 6-7.5. The cultivation time used in theinvention is set to a time longer than that required for the growthinitiation of the microorganisms, preferably, 10-100 hours. Theconcentration of dissolved oxygen (DO) in nutrient culture media is notspecifically restricted, but usually it is set to a level in the rangeof 0.5-20 ppm. The concentration of DO can be kept within the range bycontrolling aeration, stirring and aeration with oxygen, and/orincreasing the inner pressure of fermenters. The cultivation is carriedout batchwise or in continuous manner.

After completion of the cultivation of microorganisms, the presentAMYLASE is collected from the resultant cultures. When the activity ofthe present AMYLASE is present in the resultant nutrient culture mediawhich can be used intact as a crude enzyme. Conventional liquid-solidseparation methods can be employed in the invention as a method forseparating microorganisms from nutrient culture media. For example,methods for directly centrifuging cultures, filtrating cultures withprecoat filters, and separating microorganisms by membrane filtrationusing plain filters or follow fibers can be suitably used. Cultures freeof microorganisms can be used as a crude enzyme, and, preferably, theycan be concentrated by conventional manner prior to use: For example,salting out using ammonium sulfate, sedimentation using acetone andalcohol, and concentration using membranes such as plain filters andfollow fibers can be used.

Cultures free of microorganisms and concentrates thereof can beimmobilized by conventional methods. Examples of such conventionalmethods are conjugation methods using ion exchangers, covalent bonding-and absorption-methods using resins and membranes, and inclusion methodsusing high-molecular weight substances.

Crude enzymes can be used without any further treatment or may bepurified in usual manner. For example, an enzyme exhibiting a singleband on electrophoresis can be prepared by dialyzing crude enzymepreparations which had been prepared by salting out cultures to removemicroorganisms with ammonium sulfate and concentrating the resultantsolutions. The concentrate was dialyzed, and successively purified onanion-exchange column chromatography using "DEAE TOYOPEARL®",commercialized by Tosoh Corporation, Tokyo, Japan, an anion exchangeresin; hydrophobic column chromatography using "BUTYL TOYOPEARL®",commercialized by Tosoh Corporation as described above, a hydrophobicresin; and column chromatography using "PHENYL SUPERROSE® HR5/5",commercialized by Pharmacia LKB Biotechnology AB, Uppsala, Sweden.

The present AMYLASE thus obtained has the following physicochemicalproperties of:

(1) Acting

Mainly forming maltohexaose and maltoheptaose when acts on starch, butnot substantially hydrolyzing lower molecular oligosaccharides thanmaltohexaose and not substantially acting on maltoheptaose;

(2) Molecular weight

43,000±3,000 daltons on sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE);

(3) Isoelectric point (pI)

7.6±0.5 on isoelectrophoresis using ampholyte;

(4) Optimum pH

About 5.0 in the presence of calcium ion;

(5) Optimum temperature

About 70° C. in the presence of calcium ion;

(6) pH Stability

Stable at a pH in the range of about 4.5-10.5 in the presence of calciumion;

(7) Thermal stability

Stable up to a temperature of about 60° C. in the presence of calciumion;

(8) Promotion of activity and stabilization

Being promoted its activity and stabilized by calcium ion;

(9) Inhibition

Being inhibited by copper ion, lead ion, zinc ion, mercury ion and EDTA.

The activity of the present AMYLASE is assayed as follows: 0.2 ml of anenzyme solution is added to 5 ml of 0.3 w/v % soluble starch in 20 mMphosphate buffer (pH 5.5) containing 1 mM CaCl₂), and the mixturesolution is incubated at 40° C. for 10 min, followed by sampling 0.5 mlof the resultant solution, and adding the solution to 15 ml of 0.02 Nsulfate solution to suspend the enzymatic reaction. The resultant ismixed with 0.2 ml of 0.1 N iodine solution, stirred, allowed to stand at25° C. for 15 min, and determined its absorbance at a wave length of 660nm. One unit activity of the present AMYLASE is defined as the amount ofenzyme which diminishes the iodine coloration of 15 mg soluble starchwhen reacted at 40° C. for 10 min.

When used the present AMYLASE to produce saccharide compositionscontaining maltohexaose and/or maltoheptaose, it is satisfactorilyallowed to act on amylaceous substances as a substrate such as starch,amylopectin, amylose and starch hydrolyzates with an industrial viewpoint. If necessary, in the case of producing foods and beveragescontaining amylaceous substances, the present AMYLASE can be allowed toact on such foods and beverages to form therein maltohexaose and/ormaltoheptaose to prevent the retrogradation of the amylaceous substancesand to prolong the shelf-life thereof.

The present AMYLASE is added to a solution, which contains about 5-45%amylaceous substances, and, preferably, further contains about 0.5-50 mMcalcium salts such as calcium chloride in an amount of 0.5-20 units/gamylaceous substance, d.s.b., followed by the enzymatic reaction at a pHof 3-8 and a temperature of 40-90 for 1-100 hours.

To obtain the highest possible content of maltohexaose and maltoheptaosein reaction mixtures, it is desirable to allow the present AMYLASE toact on starch hydrolysates which were carefully prepared with acids orα-amylase to keep the liquefaction as low as possible, i.e. a DE lowerthan 10, preferably, a DE lower than 5.

In the present enzymatic reaction, the saccharide compositionscomprising maltohexaose and maltoheptaose, and their contents arereadily varied and increased by using the present AMYLASE and otheramylaceous substance-related enzyme(s) such ascyclomaltodextrin-glucanotransferase (EC 2.4.1.19), α-amylase, (EC3.2.1.1), β-amylase (EC 3.2.1.2), glucoamylase (EC 3.2.1.3),α-glucosidase (EC 3.2.1.20), pullulanase (EC 3.2.1.41), isoamylase (EC3.2.1.68) and maltotetraose-forming amylase (EC 3.2.1.60).

To increase the yield of maltohexaose and maltoheptaose is readilyfeasible by subjecting amylaceous substances to the action of thepresent AMYLASE together with pullulanase and isoamylase. Maltohexaoseand maltoheptaose can be arbitrarily obtained by using the resultantsaccharide compositions as a material which contain about 30-50%, d.s.b,of maltohexaose and maltoheptaose in total, and by separating theimpurities of saccharides and dextrin.

As an example of the separation methods usable in the invention, amethod using a semipermeable membrane as disclosed in Japanese PatentLaid-Open No.4,647/73, a method using a precipitant as disclosed inJapanese Patent Laid-Open No.102,854/74, and a method using astrong-acid cation exchange resin as disclosed in Japanese PatentLaid-Open Nos.148,794/84 can be arbitrarily used. These methods enablethe preparation of high maltohexaose content saccharides and highmaltoheptaose content saccharides with a purity of 90% or higher, d.s.b.Especially, methods using strong-acid cation exchange resins can beadvantageously used in an industrial scale to remove concomitantsaccharides and to obtain high maltohexaose and/or maltoheptaose contentsolutions. In this case, any one of fixed bed-, moving bed- and semimoving-methods can be employed.

Reaction mixtures after enzymatic reaction or solutions prepared byremoving concomitant saccharides from the reaction mixtures are in usualmanner subjected to filtration and centrifugation to remove insolublesubstances, and the resultant solutions are decolored with an activatedcharcoal, desalted with ion exchangers in H- and OH-form, andconcentrated into syrupy products. If necessary, the syrupy products canbe readily dried into powdery products by spray-drying method, etc.

The resultant saccharide compositions, containing maltohexaose and/ormaltoheptaose, according to the invention usually contain maltohexaoseand/or maltoheptaose in an amount of 30% or higher, and, preferably, 40%or higher, d.s.b. Although the properties of the powdery products arevaried dependently on the content of maltohexaose and/or maltoheptaose,the products have a substantial non-hygroscopicity, non-solidificationand free-flowing ability, and because of these the material- andlabor-costs for packaging, transportation and storage can be cut by alarge margin.

The powdery products thus obtained have a substantial non-hygroscopicityand a relatively-high thermal tolerance and stability, and because ofthese they can be arbitrarily used as a filler, excipient, adjuvant andpowdery base in a powdery mixed sweetener, instant juice mix, instantsoup mix, granule and tablet which have been very difficult to prepare.The powdery products can be also used in an instant pudding mix, instanthot cake mix, confectionery material, bread material and cereal materialby substituting them for the part of or the whole of products in powderform such as wheat, corn grits and starch. Saccharide compositionscontaining maltohexaitol and/or maltoheptaitol with asatisfactorily-high chemical stability can be arbitrarily prepared byhydrogenating saccharide compositions containing maltohexaose and/ormaltoheptaose.

For example, a saccharide composition containing maltohexaose andmaltoheptaose is prepared into a solution having a concentration ofabout 40-60%, d.s.b., and the solution is subjected to autoclave,admixed with Raney nickel as a catalyst in an amount of about 8-10%,d.s.b., and heated up to a temperature of 90-140° C. while stirring,followed by increasing the hydrogen pressure up to 20-150 kg/cm² toterminate the hydrogenation. Thereafter, the raney nickel is removed,and, similarly as in the preparation of saccharide compositionscontaining maltohexaose and/or maltoheptaose, decolored, desalted,purified and concentrated into syrupy products. If necessary, the syrupyproducts can be spray-dried into powdery products.

The resultant saccharide compositions containing maltohexaitol andmaltoheptaitol usually contain maltohexaitol and maltoheptaitol in anamount not less than 30%, d.s.b. The maltohexaitol and/or maltoheptaitolwith a high purity can be readily obtained by fractionation, similarlyas in the preparation of maltohexaose and maltoheptaose, comprisingremoving concomitant saccharides from saccharide compositions containingmaltohexaitol and/or maltoheptaitol as a material.

The above-mentioned saccharide compositions containing maltohexaoseand/or maltoheptaose, or those containing maltohexaitol and/ormaltoheptaitol can be arbitrarily used as a low sweetener, filler,viscosity-controlling agent, moisture-retaining agent, gloss-impartingagent, flavor-preserving agent, crystallization-preventing agent,stickiness-preventing agent for candy, and starchretrogradation-preventing agent. The saccharide compositions can bearbitrarily used in foods, beverages, feeds, cosmetics, pharmaceuticalsand shaped products as an energy-supplementing agent, and also used in avariety of compositions such as household commodities, materials foragriculture and forestry, chemical reagents, and materials for chemicalindustry.

Although the saccharide compositions containing maltohexaose and/ormaltoheptaose or those containing maltohexaitol and/or maltoheptaitolhave a relatively-low sweetness, they can be used intact as a seasoningfor sweetening. If necessary, they can be used together with adequateamounts of one or more other sweeteners, for example, powdered syrup,glucose, maltose, sucrose, isomerized sugar, honey, maple sugar,sorbitol, maltitol, lactitol, dihydrocharcone, stevioside, α-glycosylstevioside, rebaudioside, glycyrrhizin, L-aspartyl L-phenylalaninemethyl ester, saccharin, glycine and alanine; and/or a filler such asdextrin, starch or lactose.

The saccharide compositions containing maltohexaose and/or maltoheptaoseor those containing maltohexaitol and/or maltoheptaitol have thefollowing features: (i) They have a sweetness which well harmonizes withother materials having sour-, acid-, salty-, bitter-, astringent- anddelicious-tastes; and (ii) they are highly acid- and heat-resistant.Thus, they can be favorably used in food products in general as asweetener, taste-improving agent or quality-improving agent.

The saccharide compositions containing maltohexaose and/or maltoheptaoseor those containing maltohexaitol and/or maltoheptaitol can be used inseasonings such as soy sauce, powdered soy sauce, "miso","funmatsu-miso" (a powdered miso), "moromi" (a refined sake), "hishio"(a refined soy sauce), "furikake" (a seasoned fish meal), mayonnaise,dressing, vinegar, "sanbai-zu" (a sauce of sugar, soy sauce andvinegar), "funmatsu-sushi-su" (powdered vinegar for sushi),"chuka-no-moto" (an instant mix for Chinese dish), "tentsuyu" (a saucefor Japanese deep-fat fried food), "mentsuyu" (a sauce for Japanesevermicelli), sauce, catsup, "takuan-zuke-no-moto" (a premix for pickledradish), "hakusai-zuke-no-moto" (a premix for fresh white rape pickles),"yakiniku-no-tare" (a sauce for Japanese grilled meat), curry roux,instant stew mix, instant soup mix, "dashi-no-moto" (an instant stockmix), nucleic acid condiments, mixed seasoning, "mirin" (a sweet sake),"shin-mirin" (a synthetic mirin), table sugar and coffee sugar. Also,the saccharide containing maltohexaose and/or maltoheptaose or thesaccharide containing maltohexaitol and/or maltoheptaitol can be freelyused for sweetening "wagashi" (Japanese cake) such as "senbei" (a ricecracker), "arare-mochi" (a rice-cake cube), "okoshi" (a millet-and -ricecake), "mochi" (a rice paste), "manju" (a bun with a bean-jam), "uiro"(a sweet rice jelly), "an" (a bean jam), "yokan" (a sweet jelly ofbeans), "mizu-yokan" (a soft adzuki-bean jelly), "kingyoku" (a kind ofyokan), jelly, pao de Castella and "amedama" (a Japanese toffee);confectioneries such as bun, biscuit, cracker, cookie, pie, pudding,butter cream, custard cream, cream puff, waffle, sponge cake, doughnut,chocolate, chewing gum, caramel and candy; frozen desserts such as icecream and sherbet; syrups such as "kajitsu-no-syrup-zuke" (a preservedfruit) and "korimitsu" (a sugar syrup for shaved ice); pastes such asflour paste, peanut paste, fruit paste and spread; processed fruits andvegetables such as jam, marmalade, "syrup-zuke" (fruit pickles) and"toka" (conserves); pickles and pickled products such as "fukujin-zuke"(red colored radish pickles), "bettara-zuke" (a kind of whole freshradish pickles), "senmai-zuke" (a kind of sliced fresh radish pickles)and "rakkyo-zuke" (pickled shallots); meat products such as ham andsausage; products of fish meat such as fish ham, fish sausage,"kamaboko" (a steamed fish paste), "chikuwa" (a kind of fish paste) and"tenpura" (a Japanese deep-fat fried fish paste); "chinmi" (relish) suchas "uni-no-shiokara" (salted guts of sea urchin), "ika-no-shiokara"(salted guts of squid), "su-konbu" (processed tangle), "saki-surume"(dried squid strips) and "fugu-no-mirin-boshi" (a dried mirin-seasonedswellfish); "tsukudani" (foods boiled down in soy sauce) such as thoseof laver, edible wild plants, dried squid, fish and shellfish; dailydishes such as "nimame" (cooked beans), potato salad and "konbu-maki" (atangle roll); milk products; canned and bottled products such as thoseof meat, fish meat, fruit and vegetable; alcoholic beverages such asrefined sake, synthetic sake, liqueur and foreign wines; soft drinkssuch as tea, coffee, cocoa, juice, carbonated beverage, sour milkbeverage and beverage containing a lactic acid bacterium; instant hotcake mix and "sokuseki-shiruko" (an instant mix of adzuki-bean soup withrice cake), and instant soup mix; and beverages such as baby foods,foods for therapy, beverages supplemented with nutrition, peptide foodsand frozen foods; as well as for improving the tastes and qualities ofthe aforementioned food-products.

The present saccharide compositions containing maltohexaose and/ormaltoheptaose or those containing maltohexaitol and/or maltoheptaitolcan be also used in feeds and pet foods for domestic animals, poultryand fishes to improve their taste preferences. These saccharidecompositions can be arbitrarily used as a sweetener, taste-improvingagent or quality-improving agent in other products in the form of asolid, paste or liquid such as a tobacco, cigarette, dentifrice,lipstick, rouge, lip cream, internal medicine, tablet, troche, cod liveroil in the form of drop, cachou, oral refrigerant, gargle, cosmetics orpharmaceutical.

The present saccharide compositions containing maltohexaose and/ormaltoheptaose or those containing maltohexaitol and/or maltoheptaitolcan be used as a quality-improving agent or a stabilizer forbiologically active substances susceptible to lose their effectiveingredients and activities, as well as in health foods andpharmaceuticals containing biologically active substances. Examples ofsuch biologically active substances are lymphokines such as α-, β- andγ-interferons, tumor necrosis factor-α (TNF-α), tumor necrosis factor-β(TNF-β), macrophage migration inhibitory factor and interleukin 2;hormones such as insulin, growth hormone, prolactin, erythropoietin,follicle-stimulating hormone and placental hormone; biologicalpreparations such as BCG vaccine, Japanese encephalitis vaccine, measlesvaccine, live polio vaccine, smallpox vaccine, tetanus toxoid,Antivenenum Trimeresurus flavoviridis and human immunoglobulin;antibiotics such as penicillin, erythromycin, chloramphenicol,tetracycline, streptomycin and kanamycin sulfate; vitamins such asthiamine, riboflavin, L-ascorbic acid, cod liver oil, carotenoid,ergosterol and tocopherol; enzymes such as lipase, elastase, urokinase,protease, β-amylase, isoamylase, glucanase and lactose; extracts such asginseng extract, snapping turtle extract, chlorella extract, aloeextract and propolis extract; viable microorganisms such as viruses,lactic acid bacteria and yeasts; and other biologically activesubstances such as royal jelly. By using the present saccharidecompositions containing maltohexaose and/or maltoheptaose or thosecontaining maltohexaitol and/or maltoheptaitol, the aforementionedbiologically active substances are arbitrarily prepared into healthfoods and pharmaceuticals with a satisfactorily-high stability andquality without fear of losing or inactivating their effectiveingredients and activities.

As is described above, the saccharide compositions in the presentinvention can be arbitrary used in orally or parenterally administrablefoods, beverages, cosmetics and pharmaceuticals, as well as in householdcommodities, materials for agriculture and forestry, chemical reagents,and materials for chemical industry.

Methods to incorporate into above-mentioned products the presentsaccharide compositions containing maltohexaose and/or maltoheptaose orthose containing maltohexaitol and/or maltoheptaitol are conventionalones, for example, mixing, dissolving, soaking, permeating, sprinkling,applying, spraying, injecting and solidifying as long as theincorporation is attained before completion of their processings.Although the amount of these saccharide compositions to be incorporatedinto is varied dependently on the compositions used, it is usually 0.1%or higher, preferably, 0.5% or higher with respect to maltohexaose,maltoheptaose, maltohexaitol and maltoheptaitol, d.s.b. The followingexperiments explain the present invention in more detail:

Experiment 1

Production of the enzyme

A liquid nutrient culture medium, consisting of 2.0 w/v % solublestarch, 0.5 w/v % peptone, 0.1 w/v % yeast extract, 0.1 w/v % potassiumdiphosphate, 0.06 w/v % sodium phosphate dihydrate, 0.05 w/v % magnesiumsulfate heptahydrate, 0.5 w/v % calcium carbonate and water, was placedin 500-ml Erlenmeyer flasks about 100-ml aliquots, and autoclaved at121° C. for 15 min to effect sterilization, cooled, inoculated with astock culture of Alcaligenes latus D2271 (FERM BP-4578), and incubatedat 37° C. for 20 hours under stirring and shaking conditions of 200 rpm.The resultant cultures were pooled and used as a seed culture.

About 20 L of a fresh preparation of the same nutrient culture mediumused in the above culture was placed in a 30 L fermenter, sterilized,cooled to 37° C., inoculated with one v/v % of the seed culture, andincubated for about 24 hours while stirring under aerobic conditions at37° C. About 19 L of the culture was centrifuged to obtain about 18 L ofculture supernatant. The AMYLASE activity of the resultant culturesupernatant was about 58 units/ml.

Experiment 2

Purification of enzyme

The culture supernatant obtained in Experiment 1 was subjected toUF-membrane filtration to obtain about 800 ml of the concentrated enzymesolution having 1,200 units/ml of the AMYLASE. Three hundred ml of theresultant enzyme concentrate was dialyzed against 10 mM Tris-HCl buffer(pH 8.0) containing 5 mM calcium chloride for 24 hours, and centrifugedto remove insoluble substances. Four hundred ml of the resultantdialyzed supernatant was subjected to column chromatography using acolumn packed with 100 ml of "DEAE-TOYOPEARL® 650", an ion-exchanger.

The present AMYLASE did not adsorb on the ion-exchanger, and theactivity was found in non-adsorbed fractions. The fractions having theobjective enzyme activity were pooled, and dialyzed against a freshpreparation of the same buffer containing 0.5 M ammonium sulfate. Thedialyzed solution thus obtained was centrifuged to remove insolublesubstances, and the resultant supernatant was subjected to hydrophobiccolumn chromatography using a column packed with 100 ml of"BUTYL-TOYOPEARL® 650", a hydrophobic gel. The enzyme adsorbed on thegel was eluted from the column with a linear gradient buffer raging from0.5 M to 0 M, followed by recovering fractions with the enzyme activity.The resultant fractions were subje6cted to hydrophobic columnchromatography using "PHENYL SUPERROSE® HR5/5", followed by recoveringfractions with the enzyme activity.

The yield of the purified enzyme preparation obtained in the above wasabout 25% with respect to the culture supernatant. The specific activityof the purified enzyme preparation was about 1,860 units/mg protein. Theprotein was determined for its quantity by the Lowry method using bovineserum albumin as a standard. The purified enzyme preparation wasdetermined for its purity on electrophoresis using 7.5 w/v %polyacrylamide gel to exhibit a single protein band, and this revealedthat the preparation was an electrophoretically homogeneous enzyme witha relatively-high purity.

Experiment 3

Property of enzyme

The AMYLASE preparation obtained in Experiment 3 was subjected toelectrophoresis using 10 w/v % sodium dodecyl sulfate polyacrylamidegel, and this revealed that the molecular weight was about 43,000±3,000daltons in comparison with those of marker proteins commercialized byJapan Bio-Rad Laboratories, Tokyo, Japan.

The purified AMYLASE preparation was subjected to isoelectrophoresisusing polyacrylamide gel containing 2 w/v % "AMPHOLINE", an ampholytecommercialized by Pharmacia LKB Biotechnology AB, Uppsala, Sweden. Theresultant gel was determined for its pH to reveal that the enzyme has apI of about 7.6±0.5.

Effects of temperature and pH on the enzyme activity were studied in thepresence of calcium chloride in accordance with the assay as used fordetermining the enzyme activity. These results were respectively shownin FIGS. 1 and 2. The optimum temperature was about 70° C., and theoptimum pH was about 5.0. The thermal stability of the enzyme wasdetermined by incubating it in 20 mM acetate buffer containing 5 mMcalcium chloride (pH 6.0) at different temperatures for 60 min, coolingthe buffers, and assaying the remaining enzyme activity in each buffer.The pH stability of the enzyme was determined by incubating it in 20 mMacetate buffer having different pHs at 40° C. for one hour, adjustingthe buffer to pH 6.0, and assaying the remaining enzyme activity in eachbuffer. The results of thermal- and pH-stabilities were respectivelyshown in FIGS. 3 and 4. The enzyme was stable up to a temperature ofabout 60° C. and at a pH of about 4.5-10.5. The enzyme was an amylasewhich does not exhibit a starch-hydrolyzing activity, but has arelatively-high dependency on calcium ion in solutions containingsubstrates. The enzyme activity was inhibited in the presence of one mMcopper ion, lead ion, zinc ion or mercury ion, or by 10 mM EDTA.

Experiment 4

Activity on starch

To soluble starch having a final concentration of 2 w/v % in 20 mMacetate buffer (pH 5.5) containing 5 mM calcium chloride was added 50units/g starch of the purified AMYLASE obtained in Experiment 2, and themixture solution was subjected to an enzymatic reaction at 40° C.,followed by sampling the reaction solution at a prescribed time intervaland heating the samples at 100° C. for 10 min to inactivate theremaining enzyme. The resultant reaction solution was analyzed for itshydrolysis rate, blue value and saccharide composition.

The hydrolysis rate was determined by quantifying the total sugarcontent of the reaction mixture with the anthrone-sulfuric acid reactionmethod, quantifying the reducing sugar content of the reaction mixturein terms of glucose with the Somogyi-Nelson's method, and calculatingthe ratio of the reducing sugar content against the total sugar content.The blue value was determined by adding 0.1 ml of the reaction mixtureto 15 ml of 0.05 N aqueous sulfuric acid solution, mixing the resultant,adding 0.2 ml of 0.1 N iodine solution to the resultant solution todevelop color, determining the relative absorbance at 660 nm for aniodine color degree, and calculating the percentage of the iodine colordegree of each reaction solution at each reaction time to the initialiodine color degree at a reaction time 0.

The saccharide composition was determined by first desalting thereaction mixture, then analyzing the desalted solution onhigh-performance liquid chromatography (abbreviated as "HPLC"hereinafter). The system and conditions used in the analysis were CCPDsystem, an apparatus for HPLC commercialized by Tosoh Corporation,Tokyo, Japan, "MCI GEL CK04SS (10 mm.o slashed.×200 mm), a column foranalysis commercialized by Mitsubishi Chemical Corporation, Tokyo,Japan, a flow rate of 0.2 ml/min as an eluant, and a differentialrefractometer as an detector. The results were as shown in Tables 1 and2.

                  TABLE 1                                                         ______________________________________                                        Reaction time Hydrolysis rate                                                                          Iodine color                                         (min)         (%)        degree (%)                                           ______________________________________                                         0            1.9        100                                                   5            3.6        45                                                   10            5.3        19                                                   20            8.0        4.5                                                  30            9.8        2.0                                                  60            13.4       1.0                                                  90            14.7       0.9                                                  120           15.7       0.9                                                  ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Reaction                                                                              Saccharide composition (%)                                            time (min)                                                                            G1    G2      G3  G4   G5  G6     G7   ≧G8                     ______________________________________                                         5      0.0   0.0     0.2 0.2  0.3 2.1    2.2  95.0                           10      0.0   0.7     0.9 0.7  1.0 3.9    4.0  88.8                           20      0.0   1.5     2.0 1.8  1.8 7.9    7.8  77.2                           30      0.0   2.7     3.3 2.7  2.6 11.7   11.0 66.0                           60      0.0   6.0     6.0 4.3  3.8 19.4   16.1 44.4                           90      0.4   8.5     7.1 4.8  4.2 25.4   18.8 30.8                           120     0.7   10.1    8.4 4.5  4.0 25.7   19.3 27.3                           ______________________________________                                    

As is evident from the results in Table 1, it was revealed that thepresent AMYLASE was an endo-type amylase because the level of thereduction rate was larger than that of the increment of the hydrolysisrate of starch, and because lower molecular weight oligosaccharides suchas maltose and maltotriose were found from the initiation of theenzymatic reaction even if the amount was low. More particularly, theAMYLASE was revealed to be an endo-type α-amylase based on the result ofthe finding that the anomer type of the formed products was α-form whenexamined on the optical rotation of anomer type.

As is evident from the results in Table 2, it was revealed that thepresent starch-hydrolyzing enzyme is a novel α-amylase, a type of enzymewhich inherently forms maltohexaose and maltoheptaose when acts onstarch, because of the fact that it formed a larger amount ofmaltohexaose and maltoheptaose than other oligosaccharides from theinitiation of the enzymatic reaction while increasing the formationlevel as the enzymatic reaction proceeded and not decreasing theformation level even if the enzymatic reaction was prolonged. The totalyield of maltohexaose and maltoheptaose from starch was about 45%,d.s.b.

Experiment 5

Substrate specificity

Fifty units/g substrate, d.s.b., of a purified present AMYLASE obtainedby the method in Experiment 2 was added to a substrate with a finalconcentration of 2 w/v % amylose, amylopectin, glycogen, α-cyclodextrin,β-cyclodextrin, γ-cyclodextrin, pullulan, dextran, glucose, maltose,maltotriose, maltotetraose, maltopentaose, maltohexaose ormaltoheptaose, and the mixture was allowed to react at 40° C. and pH 5.5for 2 hours in the presence of 5 mM calcium chloride. The reactionmixtures before and after the enzymatic reaction were subjected to thinlayer chromatography (abbreviated as "TLC" hereinafter) using "KIESELGEL 60", an aluminum plate (20×20 cm) commercialized by E. Merck,Darmstadt, Germany, to examine whether the enzyme acted on thesaccharides as a substrate. TLC was developed once at ambienttemperature by using a developing solvent of 1-butanol:pyridine:water=6:4:1 by volume. The development of colorization waseffected by spraying to the plates a solution of 20 v/v % sulfuric acidin methanol and heating the plates at 110° C. for 10 min.

As a result, it was found that the present AMYLASE mainly formedmaltohexaose and maltoheptaose when acted on amylose, amylopectin andglycogen as a substrate similarly as in soluble starch, butsubstantially did not act on maltoheptaose even though it slightlyformed glucose and maltohexaose as a hydrolysate, and did not act onα-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, pullulan, dextran,glucose, maltose, maltotriose, maltotetraose, maltopentaose andmaltohexaose.

Experiment 6

Acute toxicity test

A powdery saccharide product rich in maltohexaose or maltoheptaose whichwas obtained by the method in Example A-7, and a syrupy product rich inmaltohexaitol or maltoheptaitol which was obtained by the method inExample A-8 were respectively administered to mice orally for theiracute toxicity test. As a result, it was revealed that these saccharideswere relatively-low in toxicity and no mouse died even when administeredwith the highest possible dose. Therefore, though not so accurate, thevalue of LD₅₀ of the saccharides was 50 g/kg or higher.

The following Examples A illustrate the preparation of the presentAMYLASE, saccharide compositions containing maltohexaose and/ormaltoheptaose prepared by using the AMYLASE, and preparation ofsaccharide compositions containing maltohexaitol and/or maltoheptaitol.Examples B illustrate compositions containing one or more of thesesaccharides.

EXAMPLE A-1

In accordance with the method in Experiment 1, a seed culture ofAlcaligenes latus D2271 (FERM BP-4578) was inoculated in a freshpreparation of the same nutrient culture medium as used in Experiment 1and cultured in a fermenter for 30 hours under agitation-aerationconditions except for culturing the microorganism at 30° C. The activityof the present AMYLASE in the resultant supernatant was 66 units/ml ofthe culture. The culture was filtered with an MF-membrane, and thefiltrate was concentrated with a UF-membrane to obtain a concentratedenzyme solution containing about 1,300 units/ml of the present AMYLASEin a yield of about 82% against the activity of the initial culture.

EXAMPLE A-2

Five % suspension (pH 5.5) of potato starch, d.s.b, was gelatinized byheating, cooled to 70° C., admixed with calcium chloride to give 0.02%,mixed with 2 units/g starch of the AMYLASE in Example A-1, liquefied andsaccharified for 20 hours. The resultant mixture was autoclaved at 120°C. for 20 min to inactivate the remaining enzyme, cooled, decolored andfiltered by an active charcoal, desalted with ion-exchange resins in H-and OH-form, purified and concentrated into a 75% syrup in a yield ofabout 95% to the material, d.s.b.

The product contained 24.2% maltohexaose and 17.7% maltoheptaose, d.s.b,and had a sweetening power of about 15% of that of sucrose. The productcan be arbitrarily used in a variety of compositions such as foods,beverages, cosmetics, pharmaceuticals and shaped products as a lowsweetener, shape-imparting agent, viscosity-controlling agent,moisture-retaining agent, gloss-imparting agent, adhesive,flavor-imparting agent, crystallization-preventing agent, andstickiness-preventing agent for candy.

EXAMPLE A-3

Ten % suspension (pH 4.5) of potato starch, d.s.b, was gelatinized byheating, cooled to 50° C., mixed with 1,000 units/g starch of isoamylasecommercialized by Hayashibara Biochemical Laboratories, Inc., Okayama,Japan, and enzymatically reacted for 20 hours. The resultant mixture wasadjusted to pH 5.2, autoclaved at 120° C. for 10 min, cooled to 70° C.,admixed with calcium chloride to give a concentration of 0.02%, d.s.b,admixed with 5 units/g starch, d.s.b., of the present AMYLASE preparedin Example A-1, liquefied and saccharified for 8 hours. The reactionsolution was autoclaved at 120° C. for 20 min, cooled, and, similarly asin the Example A-2, purified and concentrated to obtain a syrupy productwith a concentration of 75% in a yield of about 93% to the material,d.s.b. The saccharide composition consisted of 40.3% oligosaccharideshaving a degree of glucose polymerization of 5 or lower, 27.3%maltohexaose, 19.2% maltoheptaose, and dextrin having a degree ofglucose polymerization of 8 or higher 13.2%, d.s.b. Similarly as theproduct in Example A-2, the product can be arbitrarily used as asweetener with a relatively-low sweetness and relatively-low sugarcontent in a variety of compositions such as foods, beverages,cosmetics, pharmaceuticals and shaped products.

EXAMPLE A-4

Thirty-three % suspension of corn starch, d.s.b., was mixed with calciumcarbonate to give a final concentration of 0.1%, d.s.b., and theresultant mixture was adjusted to pH 6.0, admixed with 0.2% per gstarch, d.s.b., of "TERMAMYL® 60 L", α-amylase commercialized by NovoIndustri A/S Copenhagen Denmark, and subjected to an enzymatic reactionat 95° C. for 15 min. The reaction mixture was autoclaved at 120° C. for30 min, cooled to 50° C., admixed with 500 units/g starch of isoamylasecommercialized by Hayashibara Biochemical Laboratories Inc., Okayama,Japan, and 1.8 units/g starch of the present AMYLASE prepared in theExample A-1, and subjected to an enzymatic reaction for 40 hours. Thereaction mixture was similarly as in Example A-2 heated, cooled,purified, concentrated and spray-dried to obtain a powdery compositioncontaining maltohexaose and maltoheptaose having a moisture contentlower than 2% in a yield of about 90%, d.s.b.

The saccharide composition consisted of 38.8% oligosaccharides having adegree of glucose polymerization of 5 or lower, 26.7% maltohexaose,15.7% maltoheptaose, and 18.8% dextrin having a degree of glucosepolymerization of 8 or higher. Similarly as the product in Example A-2,the product can be arbitrarily used as a sweetener with a relatively-lowsweetness and relatively-low sugar content in a variety of compositionssuch as foods, beverages, cosmetics, pharmaceuticals and shapedproducts.

EXAMPLE A-5

A saccharide composition containing maltohexaose and maltoheptaose,obtained by the method in Example A-3, was column chromatographed byusing a strong-acid cation exchange resin for fractionation to increasethe content of maltohexaose and maltoheptaose. Thus, the saccharidecomposition was concentrated into 55 w/w % solution as a materialsaccharide solution. "DIAION SK 104 (K⁺ -form, polymerization degree of4%)" a resin commercialized by Mitsubishi Chemical Corporation, Tokyo,Japan, was packed in 4 jacketed-stainless steel columns having an innerdiameter of 5.4 cm, and the columns were cascaded in series to give atotal gel-bed depth of 20 m. The columns were heated to give the innercolumn temperature of 60° C., and fed with 7 v/v % of the saccharidesolution while keeping at the temperature, followed by fractionating thesaccharide solution by feeding to the columns with 60° C. hot water at aflow rate of SV (space velocity) 0.12 and collecting fractionscontaining 35% or higher of maltohexaose or 25% or higher ofmaltoheptaose. The fractions were pooled, purified and concentratedsimilarly as in Example A-2 to obtain a syrupy saccharide compositionrich in maltohexaose and maltoheptaose in a yield of about 50% to thematerial saccharide solution, d.s.b. The product contained about 45%maltohexaose and about 31% maltoheptaose, d.s.b, and the sweeteningpower was less than 10% of that of sucrose. Similarly as in Example A-2,the product can be arbitrarily used as a sweetener with less sweetnessand sugar content in a variety of compositions such as foods, beverages,cosmetics, pharmaceuticals and shaped products.

EXAMPLE A-6

A syrupy saccharide composition rich in maltohexaose and maltoheptaose,obtained by the method in Example A-5, was spray-dried to obtain apowdery saccharide composition with a moisture content less than about2% and rich in maltohexaose and maltoheptaose in a yield of about 98%syrup, d.s.b. Similarly as in Example A-2, the product can bearbitrarily used as a sweetener with less sweetness and sugar content ina variety of compositions such as foods, beverages, cosmetics,pharmaceuticals and shaped products.

EXAMPLE A-7

A saccharide composition rich in maltohexaose and maltoheptaose,obtained by the method in Example A-5, was prepared into a 50% solutionwhich was used as a material saccharide solution. "DIAION SK 104 (K⁺-form, polymerization degree of 4%)", a strong-acid cation exchangercommercialized by Mitsubishi Chemical Corporation, Tokyo, Japan, wasused as a resin for fractionation and packed similarly as in Example A-5to column. The column was heated to give the inner column temperature of60° C., and fed with 5 v/v % of the saccharide solution while keeping atthe temperature, and the saccharide solution was fractionated by feedingto the column with 60° C. hot water to separatory collect fractionscontaining 80% or higher of maltohexaose and fractions containing 70% orhigher of maltoheptaose, d.s.b. The fractions were respectively pooled,purified, concentrated and spray-dried into a powdery saccharidecomposition rich in maltohexaose and a powdery saccharide compositionrich in maltoheptaose having a moisture content less than about 2% in ayield of about 30% and about 20%, d.s.b., respectively.

The purities of the powdery saccharide composition rich in maltohexaoseand the powdery saccharide composition rich in maltoheptaose were about89% and about 81%, d.s.b., and their sweetening powers were less than10% of that of sucrose. Similarly as in Example A-2, the products can bearbitrarily used as a sweetener with less sweetness and sugar content ina variety of compositions such as foods, beverages, cosmetics,pharmaceuticals and shaped products.

EXAMPLE A-8

A syrupy saccharide composition containing maltohexaose andmaltoheptaose, obtained by the method of Example A-3, was prepared intoa 50% solution which was then placed in an autoclave, admixed with 10%raney nickel as a catalyst, and heated up to a temperature of 90-125° C.while stirring, followed by increasing hydrogen pressure to 20-100kg/cm² to terminate hydrogenation. Thereafter, the raney nickel wasremoved from the reaction mixture, and, similarly as in Example A-2, thereaction mixture was purified and concentrated to obtain a saccharidecomposition containing maltohexaitol and maltoheptaitol having amoisture content of 25% in a yield of about 90% syrup, d.s.b.

The product contains 27% maltohexaitol, 19% maltoheptaitol, d.s.b, andthe sweetening power was about 20% of that of sucrose. The product canbe arbitrarily used in a variety of food products as a sweetener withless sweetness and sugar content, body-imparting agent,viscosity-controlling agent, moisture-retaining agent, gloss-impartingagent, adhesive, flavor-imparting agent, crystallization-preventingagent, and stickiness-preventing agent for candy.

EXAMPLE A-9

A saccharide composition rich in maltohexaose and maltoheptaose,obtained by the method in Example A-5, was hydrogenated by the method inExample A-8, and the resultant was purified and concentrated into a 50%saccharide solution rich in maltohexaitol and maltoheptaitol. Asaccharide solution thus obtained was column chromatographed inaccordance with the method in Example A-7 by using a strong-acid cationexchange resin to separatory collect fractions containing 80% or higherof maltohexaitol and fractions containing 70% or higher ofmaltoheptaitol, which were then respectively pooled, purified andconcentrated into a saccharide syrup rich in maltohexaitol and asaccharide syrup rich in maltoheptaitol, both of which had aconcentration of about 75% and obtained in yields of about 30% and about20%, d.s.b., respectively The purities of the saccharide syrups thusobtained were respectively about 90% and about 82%, d.s.b., and theirsweetening powers were less than 10% of that of sucrose. Similarly as inExample A-2, the product can be arbitrarily used as a sweetener withless sweetness and sugar content in a variety of compositions such asfoods, beverages, cosmetics, pharmaceuticals and shaped products.

EXAMPLE B-1

Sweetener

One part by weight of a syrupy saccharide composition containingmaltohexaose and maltoheptaose, obtained by the method in Example A-2,was homogeneously admixed with 0.02 parts by weight of "αG Sweet",α-glycosyl stevioside commercialized by Toyo Sugar Refining Co., Ltd.,Tokyo, Japan, to obtain a syrupy sweetener. The product had asatisfactory sweetness and a 2-fold higher sweetening power of sucrose,and the caloric value was lowered to about 1/2 of that of sucrose.Because of this, it can be suitably used as a low-caloric sweetener forlow-caloric food products for fat persons and diabetics who arerestricted to a reduced calorie intake. The product scarcely forms acidsand insoluble glucans when dental carries-inducing microorganisms act onit, and this renders it useful for sweetening food products directed toprevent dental carries.

EXAMPLE B-2

Custard cream

Five hundred parts by weight of corn starch, 400 parts by weight of apowdery saccharide composition rich in maltohexaose and maltoheptaose,500 parts by weight of maltose, and 5 parts by weight of salt werepassed through a sieve to homogeneity. The resultant mixture was admixedwith 1,400 parts by weight of egg, and gradually mixed with 5,000 partsby weight of a boiling milk. The mixture thus obtained was continuedstirring while heating, and the heating was stopped when the corn starchin the mixture was completely gelatinized to give the whole contentssemitransparent, followed by cooling the resultant and adding thereto anadequate amount of a vanilla flavor. The product has a smooth surfaceand gloss, as well as a mild taste and sweetness.

EXAMPLE B-3

Sweet rice jelly (uiro)

Ninety parts by weight of rice powder, 20 parts by weight of cornstarch, 20 parts by weight of sugar, one part by weight of a powderedgreen tea, an adequate amount of water, and 90 parts by weight of asyrupy saccharide composition containing maltohexaitol andmaltoheptaitol, obtained by the method in Experiment A-8, were kneadedto homogeneity. The resultant mixture was placed in a vessel and steamedfor 60 min to obtain a rice jelly with powdered green tea. The producthas a satisfiable gloss, biting property and flavor. The retrogradationof starch in the product is well prevented, and the shelf-life issatisfactory.

EXAMPLE B-4

Hard candy

Seventy hundred parts by weight of a syrupy saccharide compositioncontaining maltohexaose and maltoheptaose, obtained by the method inExample A-3, was mixed with 90 parts by weight of sucrose, and theresultant mixture was concentrated by heating in vacuo until themoisture content lowered to below 2%. The concentrated solution wasadmixed with 0.15 parts by weight of citric acid and adequate amounts ofa lemon flavor and a coloring agent, and the resultant mixture wasformed in usual manner to obtain the desired product. The product is ahigh-quality hard candy having a satisfactory taste and biting property,as well as having a relatively-low hygroscopicity and a less stickiness.

EXAMPLE B-5

Radish pickled in sake lees ("bettara-zuke")

Thirty kg of radish was in usual manner successively pickled with salt,saccharide, and 4 kg of a well-mixed composition consisting of one partby weight of a syrupy saccharide composition containing maltohexaose andmaltoheptaose, obtained by the method in Example A-2, 3 parts by weightof maltose, 0.05 parts by weight of a powdered licorice, 0.008 parts byweight of malic acid, 0.07 parts by weight of sodium glutamate, 0.03parts by weight of potassium sorbate and 0.2 parts by weight ofpullulan. The product exerted a satisfiable gloss and texture, and had asatisfactory sweetness and biting property.

EXAMPLE B-6

Intubation nutrition

A composition was prepared by mixing 20 parts by weight of a powderysaccharide composition containing maltohexaose and maltoheptaose,obtained by the method in Example A-4, 1.1 parts by weight of glycine,one part by weight of sodium glutamate, 0.4 parts by weight of calciumlactate, 0.1 part by weight of magnesium carbonate, 0.01 part by weightof thiamine, and 0.01 part by weight of riboflavin. Twenty-four galiquots of the composition were distributed to laminated-aluminum smallbags which were then heat sealed to obtain the captioned product. Inuse, one bag of the product is dissolved in about 33-500 ml water, andadministered to the nasal cavity or stomach by the intubation feeding asan intubation nutrition. The product can be administered to not onlyhuman but also livestock.

EXAMPLE B-7

Intubation nutrition

A composition was prepared by mixing 80 parts by weight of a powderysaccharide composition containing maltohexaose and maltoheptaose,obtained by the method in Example A-6, 190 parts by weight of driedyolk, 209 parts by weight of skim milk, 4.4 parts by weight of sodiumchloride, 1.85 parts by weight of potassium chloride, 4 parts by weightof magnesium sulfate, 0.01 part by weight of thiamine, 0.1 part byweight of sodium ascorbate, 0.6 parts by weight of vitamin E acetate and0.04 parts by weight of nicotinamide. Twenty-five g aliquots of thecomposition were distributed to laminated-aluminum small bags which werethen heat sealed to obtain the captioned product. In use, one bag of theproduct is dissolved in about 150-300 ml water, and administered as anintubation nutrition to the nasal cavity, gullet and stomach by theintubation feeding.

EXAMPLE B-8

Tablet

Four parts by weight of a powdery saccharide composition containingmaltohexaose and maltoheptaose, obtained by the method in Example A-6,was mixed to homogeneity with 50 parts by weight of aspirin, 10 parts byweight of maltose and 4 parts by weight of corn starch, and theresultant mixture was tabletted by a 20 R punch having a diameter of 12mm to obtain a tablet, 680 mg in weight, 5.25 mm in thickness and 8±1 kgin hardness. The product is a readily-swallowable tablet with anadequate sweetness.

EXAMPLE B-9

Milky lotion

A half part by weight of polyoxyethylene behenyl ether, one part byweight of polyoxyethylene sorbitol tetraoleate, one part by weight ofoil-soluble glyceryl monostearate, 0.5 parts by weight of behenylalcohol, one part by weight of avocado oil, 3.5 parts by weight of asyrupy saccharide composition rich in maltohexaitol, obtained by themethod in Example A-9, one part by weight of α-glycosyl rutin andappropriate amounts of vitamin E and antiseptic were mixed by heating inusual manner, and the mixture was mixed with 5 parts by weight of1,3-butyleneglycol, 0.1 part by weight of carboxyvinyl polymer and 85.3parts by weight of refined water. The mixture thus obtained wasemulsified by a homogenizer. The product is a moisture-retaining milkylotion which is favorably usable as a sunscreen agent or askin-whitening agent.

EXAMPLE B-10

Skin cream

Two parts by weight of polyoxyethylene glycol monostearate, 5 parts byweight of self-emulsifying glyceryl monostearate, 2 parts by weight ofα-glycosyl rutin, one part by weight of liquid paraffin, 10 parts byweight of glyceryl trioctanate, 4 parts by weight of a powderysaccharide composition containing maltohexaose and an adequate amount ofan antiseptic were dissolved by heating, and the resultant solution wasmixed with 5 parts by weight of 1,3-butylene glycol and 66 parts byweight of refined water. The resultant mixture was emulsified by ahomogenizer and admixed with an adequate amount of a flavor whilestirring to obtain a skin cream. The product is a cream with asatisfactory spreadability and suitably usable as a sunscreen cream, askin-refining agent or a skin-whitening agent.

EXAMPLE B-11

Dentifrice

Forty-five parts by weight of calcium hydrogen phosphate, 1.5 parts byweight of sodium laurate, 25 parts by weight of glycerine, 0.5 parts byweight of polyoxyethylene sorbitan laurate, 15 parts by weight of asyrupy saccharide composition rich in maltohexaose and maltoheptaose,obtained by the method in Example A-5, 0.02 parts by weight of saccharinand 0.05 parts by weight of antiseptic were mixed with 13 parts byweight of water. The product with a superior gloss and detergency issuitably used as a dentifrice.

EXAMPLE B-12

Fertilizer in the form of rod

Seventy parts by weight of a compound fertilizer comprising 14% N, 8% P₂O₅, 12% K₂ O, 5 parts by weight of pullulan, 5 parts by weight of asaccharide composition containing maltohexaose and maltoheptaose,obtained by the method in Example A-3, 15 parts by weight of calciumsulfate and 5 parts by weight of water were mixed to homogeneity, andthe resultant mixture was heated to 80° C. by an extruder under theconditions of an L/D ratio of 20, a pressure ratio of 1.8 and a diediameter of 30 mm, to obtain the captioned product. In use, the productdoes not require a special vessel for carrying, and has a readilyhandleability and a satisfactory strength for a total layer application.The elution speed of the ingredients contained in the product iscontrollable by varying the composition. If necessary, the product canbe readily admixed with one or more plant hormones, agriculturalchemicals and soil conditioners.

As is described above, the present inventors found that the presentAMYLASE has a relatively-high optimum temperature and thermal stability,as well as a relatively-wide range of optimum pH and stable pH.Furthermore, they found that the yield of the present AMYLASE producedby microorganisms is considerably high, and this renders it readily beused in the production of saccharide compositions rich in maltohexaoseand/or maltoheptaose and those rich in maltohexaitol and/ormaltoheptaitol from amylaceous substances. Thus, the present inventionhas a great significance in this field.

In addition, the saccharide compositions rich in maltohexaose and/ormaltoheptaose and their hydrogenated products can be used as a sweetenerwith less sweetness in a variety of compositions, for example, abody-imparting agent, viscosity-controlling agent, moisture-retainingagent, and gloss-imparting agent, and also used as anutrition-supplementing agent in a variety of compositions such asfoods, beverages, cosmetics, pharmaceuticals and shaped products.

What is claimed is:
 1. A saccharide composition comprising hydrogenateddextrin or non-hydrogenated dextrin having a degree of glucosepolymerization of 8 or more and at least about 30 w/w % of a memberselected from the group consisting of (i) maltohexaose andmaltoheptaose, (ii) maltohexaitol and maltoheptaitol, and (iii) mixturesthereof, wherein the proportion of said maltohexaose to maltoheptaose,and the proportion of maltohexaitol to maltoheptaitol is at least about1.2, and the amount of maltoheptaose or maltoheptaitol is at least 16.1w/w %, and the proportion of hydrogenated dextrin or non-hydrogenateddextrin having a degree of glucose polymerization of five or lower to(i) said maltohexaose and said maltoheptaose, (ii) said maltohexaitoland said maltoheptaitol, or (iii) mixtures of (i) and (ii) is not morethan about 0.91.
 2. The composition as claimed in claim 1, wherein saidmaltohexaose and/or maltoheptaose are prepared by the processcomprising:(a) allowing a maltohexaose and maltoheptaose-forming amylaseto act on starch, said maltohexaose and maltoheptaose-forming amylasehaving an activity of mainly forming maltohexaose and maltoheptaose fromstarch, but substantially not having an activity of hydrolyzingmaltohexaose and a lower molecular oligosaccharide than maltohexaose,together with or without a starch debranching enzyme to formmaltohexaose and/or maltoheptaose; and (b) collecting the resultantsaccharide composition containing maltohexaose and/or maltoheptaose. 3.The composition as claimed in claim 2, wherein said starch in the step(a) is hydrolyzed by allowing the amylase to act on a solutioncontaining about 5-45 w/w % of said starch in an amount of about 0.5-20units/g starch, on a dry solid basis, at a pH 3-8 and a temperature of40-90° C. for 1-100 hours.
 4. The composition as claimed in claim 1,wherein said maltohexaitol and/or maltoheptaitol is prepared by theprocess comprising:(a) allowing to act on starch a maltohexaose andmaltoheptaose-forming amylase which has an activity of mainly formingmaltohexaose and maltoheptaose from starch, but does not substantiallyhave an activity of hydrolyzing maltohexaose and a lower molecularoligosaccharide than maltohexaose, together with or without a starchdebranching enzyme to form maltohexaose and/or maltoheptaose; (b)hydrogenating the resultant saccharide composition containingmaltohexaose and/or maltoheptaose into maltohexaitol and/ormaltoheptaitol; and (c) collecting the resultant maltohexaitol and/ormaltoheptaitol.
 5. The composition as claimed in claim 4, wherein saidstarch in the step (a) is hydrolyzed by allowing the amylase to act on asolution containing about 5-45 w/w % of said starch in an amount ofabout 0.5-20 units/g starch, on a dry solid basis, at a pH 3-8 and atemperature of 40-90° C. for 1-100 hours.
 6. The composition as claimedin claim 4, wherein the step (b) is effected by adding an about 8-10 w/w% of the raney nickel to the resultant solution in the step (a), andheating the resultant mixture at 90-140° C. and at a hydrogen pressureof 20-150 kg/cm².
 7. The composition as claimed in claim 4, wherein thestep (b) further contains a step of removing saccharides other thanmaltohexaitol and maltoheptaitol from the resultant mixture in the step(b).
 8. The composition as claimed in claim 7, wherein the removing stepis a column chromatography using a strong-acid cation exchanger.
 9. Thecomposition of claim 1, which contains at least 0.1 w/w %, d.s.b., ofsaid maltohexaose, maltoheptaose, maltohexaitol and/or maltoheptaitol.10. The composition as claimed in claim 1, which is in the form of afood, a beverage, a cosmetic, a pharmaceutical or a shaped product.